PROJECT SUMMARY Multiple sclerosis (MS) is a life-long, debilitating disease in both males and females. Symptoms include loss of motor function, neuropathic pain, cognitive impairments, and impaired social interaction. MS is furthermore associated with elevations in circulating and central (spinal cord and brain) levels of pro- inflammatory cytokines and decreased levels of anti-inflammatory cytokines, suggesting a dysregulation of immune and glial processes resulting in chronic inflammation. This ongoing inflammation is important in demyelination, chronic glial activation, and neuronal death characteristic of the disease. Targeted suppression of spinal cord neuroinflammation using anti-inflammatory strategies dramatically improves symptoms of experimental autoimmune encephalomyelitis (EAE), a rat model of MS. Looking forward toward translation, what is needed is not our current approaches that are injected intrathecally, but rather a means to effectively treat EAE/MS via a clinically relevant, orally available, blood-brain barrier permeable small molecule that targets EAE/MS pathology driven by neuroinflammation. We have discovered, and extensively characterized, such a small molecule. This drug (the non-opioid (+)-isomer of naltrexone; (+)-naltrexone) is rapidly moving toward FDA application for Investigational New Drug status. This is a selective antagonist at toll-like receptor 4 (TLR4) and TLR2. As it fails to bind classical opioid receptors, (+)-naltrexone does not interfere with the efficacy of opioids for pain control or normal functioning of opioid receptor systems. Targeting TLR2 and TLR4 arises from an extensive literature demonstrating the importance of these receptors in the neuroinflammatory processes and EAE/MS symptoms to be studied here. A complimentary series of behavioral and immunohistochemistry studies are proposed in males and females which will explore the ability of (+)-naltrexone to suppress EAE-induced (a) paresis/paralysis, (b) neuropathic pain, (c) cognitive impairment, and (d) social interaction impairment, as well as (e) improve survival, when (+)-naltrexone is systemically administered across days, comparing dosing early vs. late in the EAE timecourse. The IHC studies will analyze brain and spinal cord tissues collected after early vs. late (+)- naltrexone treatment (mapping onto the behavioral studies) to define whether (+)-naltrexone suppresses glial activation, neuronal cell death, and demyelination, as well as stimulates remyelination, as predicted. These studies create two complimentary Aims. Aim 1 explores a novel means of positively intervening in EAE-induced motor dysfunction, neuropathic pain, deficits in social interaction and cognition, and loss of life by targeting TLR4/TLR2 by systemic administration of (+)-naltrexone. Aim 2 transitions to an initial exploration of potential mechanisms underlying pathophysiology, focusing on the impact of TLR4/TLR2 blockade on demyelination and remyelination, neuronal cell death, and neuroinflammation. These studies provide a thorough investigation of the role of TLR4/TLR2 in major MS-relevant symptoms of EAE.